The podocyte has become a crucial focus as a target for interventions in kidney disease due to its key role in regulating glomerular permeability and maintaining glomerular structure. Podocyte injury is believed to be pathogenetically and prognostically important in diabetic nephropathy (DN). One of the main factors determining pathological changes of glomerular morphology and permeability are linked to elevation of podocyte intracellular calcium ([Ca2+]i). Transient receptor potential canonical (TRPC) channels are important players in the pathogenesis of renal and cardiovascular diseases. ATP is a critical signaling molecule playing key role in podocyte function. However, our knowledge about purinergic signaling in glomeruli and their regulation of TRPC channels and [Ca2+]i in podocytes in the setting of DN is rudimentary and therefore is the focus of the current proposal. The central hypothesis of this proposal is that in diabetes significant changes in [Ca2+]i homeostasis in podocytes occur, which are mediated by: 1) increased concentration of extracellular ATP; 2) remodeling of purinergic signaling from metabotropic P2Y to ionotropic P2X receptors; 3) excessive production of ROS; and 4) hyperactivity of TRPC channels; altogether these events lead to glomeruli damage, proteinuria and, consequently, ESRD. We further hypothesize that increased [Ca2+]i influx in podocytes results in a pathological increase in glomeruli permeability to albumin. Based on the preliminary data and published findings, the main objective of this project is to define the specific mechanisms mediating the effect of ATP and ROS on TRPC channels in freshly isolated glomeruli and to identify the pharmacological targets that control glomerular albumin permeability in the pathogenesis of DN. To explore this idea, we have developed novel approaches that allow assessing ATP and H2O2 release with enzymatic biosensors ex vivo and in vivo; measuring endogenous TRPC channels activity with patch clamp in podocytes of intact glomeruli; quantifying calcium flux in freshly isolated glomeruli; studying glomerular albumin permeability ex vivo. T2DN and streptozotocin treated Dahl salt-sensitive rats will be used to test our hypotheses in models of both type 1 and type 2 diabetes. Here we will test the following Specific Aims: 1) To determine basal and Ang II-induced concentrations of extracellular ATP in diabetic animals, and to define the effects of ATP on TRPC channels function; 2) To identify the contribution of ROS in ATP driven signaling pathways; 3) To determine a consequence of altered glomerular permeability in response to extracellular ATP and TRPC-dependent calcium influx; 4) To define the contributions of specific P2 receptors by testing the effects of their inhibition on the development of diabetic nephropathy. This research while fundamental in nature will begin to fill a large gap in knowledge and impact the health and welfare of both the U.S military personnel and all beneficiaries. It is the hope, that through the described studies a better understanding of the impact of calcium homeostasis in progression of diabetic nephropathy will be realized. The research proposed in this application is novel for it is the first to directly define the role of TRPC channels and purinergic signaling in DN. This research will result in significant findings that will advance our understanding of this disease. It may determine TRPC channels or specific P2 receptors as new targets for therapeutic control of DN and move towards eradication of this disease. Thus, this application has direct relevance to the health care needs of the U.S. Veteran population and their family members.